Male Sprague-Dawley rats, aged 8 weeks and weighing 220–250 g, were procured from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). They were acclimated for one week prior to the initiation of experimental procedures. They were randomly assigned to one of 6 groups: Control, Model, Model + small interfering RNA-negative control (Model + si-NC), Model + small interfering RNA for VPO1 (Model + si-VPO1), Model + si-VPO1 + overexpressed negative control (Model + si-VPO1 + oe-NC), and Model + si-VPO1 + overexpressed CYLD vector (Model + si-VPO1 + oe-CYLD), with 5 rats per group. The Model group received weekly intraperitoneal injections of 2.0 mg/kg adriamycin (ADR, 25316-40-9, Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China) for 6 consecutive weeks to construct the CHF rat model [24]. In addition to ADR, rats in the Model + si-NC, Model + si-VPO1, Model + si-VPO1 + oe-NC, and Model + si-VPO1 + oe-CYLD groups were also administered 500 µL of 8 $\times$ 10⁵ TU of the respective lentiviral vectors: si-NC, si-VPO1, si-VPO1 + oe-NC, and si-VPO1 + oe-CYLD [25]. Throughout the experiment, the health of the animals was closely monitored, and no mortality was observed. For euthanasia, cervical dislocation was employed to ensure rapid death. All experimental procedures and animal handling were conducted in accordance with the approval of the Animal Care and Use Committee of the Affiliated Changsha Hospital of Xiangya School of Medicine, Central South University (No. 2021-69).

Rat myocardial cells, H9c2 (AW-CNR083, Abiowell, Changsha, China), were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, AW-M003, Abiowell) supplemented with 10% fetal bovine serum (10099141, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (AWH0529a, Abiowell) and maintained in an incubator at 37 °C with 5% CO₂. All cell lines were validated by STR profiling and tested negative for Mycoplasma sp.

H9c2 cells were categorized into six groups: Control, Model, Model + si-NC, Model + si-VPO1, Model + si-VPO1 + oe-NC, and Model + si-VPO1 + oe-CYLD. Cells in the Model group were treated with 0.5 µmol/L ADR for 24 h [26]. Cells in the Model + si-NC group were transfected with si-NC for 20 min following ADR treatment. For the Model + si-VPO1 group, H9c2 cells were transfected with si-VPO1 (5^′-GGGCAGAGATACAGCACATCA-3^′) for 20 min post-ADR treatment. In the Model + si-VPO1 + oe-NC and Model + si-VPO1 + oe-CYLD groups, cells were transfected with oe-NC and oe-CYLD, respectively, in addition to the si-VPO1 treatment.

Total RNA was extracted from rat myocardial tissues and H9c2 cells using the Trizol reagent (15596026, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. After converting the RNA to cDNA with the effective mRNA reverse transcription kit (CW2569, Beijing CoWin Biotech, Beijing, China), 2 µL of cDNA was used for RT-qPCR analysis on a QuantStudio1 system (ABI, Carlsbad, CA, USA). The reaction conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 seconds and 60 °C for 30 seconds. The relative mRNA levels of target genes were calculated using the 2^{-Δ⁢Δ⁢Ct} method. Specific primer sequences are detailed in Table 1.

Total protein was extracted from rat myocardial tissues and H9c2 cells. The BCA protein quantification kit (AWB0156, Abiowell) was used to determine the protein concentration. Proteins were mixed with 5$\times$ loading buffer (AWB0055, Abiowell), boiled for 5 min, and then rapidly cooled on ice. Electrophoresis was performed at a constant voltage of 75 V for 130 min. The process was halted when the bromophenol blue dye reached the bottom of the gel. Proteins were then transferred to nitrocellulose membranes using electrophoresis at 300 mA. Next, the membranes were rinsed once with 1$\times$ phosphate-buffered saline with Tween 20 (1$\times$ PBST, AWI0130a, Abiowell) and blocked by immersion in 1$\times$ PBST containing 5% skimmed milk and shaken for 90 min. Primary antibodies were incubated with the membranes overnight at 4 °C. Secondary antibodies were applied for 1.5 h at room temperature. Details of the antibodies used are provided in Table 2. After incubation, the membranes were washed three times with 1$\times$ PBST. Lastly, the enhanced chemiluminescence (ECL) solution (AWB0005, Abiowell) was applied to the membranes, and protein bands were visualized using a chemiluminescent imaging system (ChemiScope6100, Clinx Science Instruments, Shanghai, China).

To assess protein stability, proteins were treated with 50 µg/mL cycloheximide (CHX, 583794, Gentihold, Beijing, China) for 0, 1, 2, and 3 h. The treated proteins were then analyzed according to the WB procedure described above [27].

The myocardial tissue sections were first baked, placed in xylene and subjected to ethanol at concentrations of 100%, 95%, 85%, and 75%. For antigen retrieval, the sections were immersed in 0.01 mol/L citrate buffer (AWT0732a, Abiowell), microwaved to boiling, and allowed to cool for 20 min. After rinsing three times with phosphate-buffered saline (PBS, AWR0213a, Abiowell), the antigen retrieval process was completed. Sections were treated with 1% periodate (AWI0186a, Abiowell) at room temperature for 10 min to block endogenous enzyme activity, followed by three rinses with PBS. Primary antibody VPO1 (PA5-144080, 1:200, Thermo Fisher Scientific) was incubated with the sections overnight at 4 °C. Secondary antibody horseradish peroxidase (HRP) goat anti-rabbit IgG (SA00001-2, 1:1000, Proteintech, Chicago, IL, USA) was applied to the sections for 30 min at 37 °C. The sections were then treated with the diaminobenzidine (DAB) working solution (ZLI9017, Zsbio, Beijing, China) for color development. Following re-staining with hematoxylin (AWI0009a, Abiowell) and dehydration through graded alcohol (60–100%) for 5 min each, the sections were cleared in xylene. Finally, the sections were mounted and examined under a microscope (DSZ2000X, Cnmicro, Beijing, China).

Cells in the logarithmic growth phase were harvested, counted, and seeded at a density of 5 $\times$ 10³ cells/well into 96-well plates, with three replicate wells per group. Each well was filled with medium containing 10% CCK-8 (AWC0114a, Abiowell) and incubated at 37 °C with 5% CO₂. Absorbance at 450 nm was measured using an enzyme-labeled instrument (MB-530, Heleas, Shenzhen, China).

The cellular lactate dehydrogenase (LDH) level was measured using the LDH assay kit (A020-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The N-terminal prohormone of brain natriuretic peptide (NT-proBNP) level was assessed with the NT-proBNP kit (CSB-E08752r, Cusabio, Wuhan, China). Aspartate aminotransferase (AST) levels were determined using the AST assay kit (C010-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The creatine kinase-myocardial band (CK-MB) level in myocardial tissues was evaluated with the CK-MB kit (CSB-E14403r, Cusabio).

The cells were stained with 30.0 µL of fluorochrome-labeled inhibitor of pan-caspase (FLICA) staining solution (ab219935, Abcam, Cambridge, UK) for 1 h in the dark. Following staining, cells were rinsed with washing buffer (ab219935, Abcam). The cells were then mixed with propidium iodide (PI, ab219935, Abcam) and washed with PBS. Flow cytometry was performed using a flow cytometer (A00-1-1102, Beckman, Pasadena, CA, USA) to analyze the results.

Detection of protein levels: H9c2 cell sections were rinsed with PBS and fixed in 4% paraformaldehyde (AWI0056a, Abiowell). Cells were permeabilized with 0.3% Triton X-100 (AWT0107a, Abiowell) for 30 min at 37 °C, followed by blocking with 5% bovine serum albumin (BSA, AWT0206a, Abiowell) for 60 min at 37 °C. Primary antibody RIPK1 (17519-1-AP, 1:50, Proteintech) was incubated with cells overnight at 4 °C. Goat anti-rabbit IgG (H+L) secondary antibody (AWS0005c, 1:200, Abiowell) was applied for 1.5 h at 37 °C. Nuclei were stained with the DAPI working solution (AWC0291a, Abiowell). Sections were mounted with buffered glycerol (AWI0178a, Abiowell) and visualized using a fluorescence microscope (BA410T, Motic, Xiamen, China).

Wheat Germ Agglutinin (WGA) Staining: Rat myocardial tissue sections were treated with sodium borohydride solution (C189300050, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). Sections were subsequently blocked with 0.3% hydrogen peroxide (80070961, Sinopharm Chemical Reagent Co., Ltd) and 5% BSA. Primary antibody Evans blue dye (EBD, bs-1558R, 1:100, Bioss, Beijing, China) was incubated with the sections overnight at 4 °C. Goat anti-rabbit IgG (H+L) secondary antibody was applied for 30 min at 37 °C. Sections were then reacted with TYP-570 (AWI0693a, Abiowell) and stained with WGA staining solution (W21405, Thermo Fisher Scientific). Nuclei were stained with the DAPI working solution. Tissue sections were cleared with Sudan Black solution and examined under a fluorescence microscope.

Detection of Co-localization: The rats’ heart tissues were embedded and baked at 60 °C for 12 h. Sections were deparaffinized, treated with Tris-EDTA buffer (pH 9.0, AWI0117a, Abiowell), microwaved to boiling, cooled to room temperature, and washed with PBS for 3 min to complete antigen retrieval. Sections were then treated with sodium borohydride solution for 30 min at room temperature, followed by 75% ethanol for 1 min, Sudan Black B (AWI0468a, Abiowell) for 15 min, and 10% normal serum 5% BSA for 60 min. Primary antibody VPO1 (1:200) was incubated with the sections overnight at 4 °C. Secondary antibodies HRP goat anti-rabbit IgG (1:1000) were applied for 30 min at 37 °C. After rinsing with PBS, sections were incubated with TSA-520 fluorescent dye (AWI0693a, Abiowell) at 37 °C away from light. Moreover, this process was repeated using Troponin T (1:100, ABS1675, Merck KGaA, Darmstadt, Germany), Vimentin (1:100, 10366-1-AP, Proteintech), or platelet/endothelial cell adhesion molecule-1 (CD31, 1:100, 11265-1-AP, Proteintech) as primary antibodies and TSA-570 (AWI0693a, Abiowell) as the fluorescent dye. Nuclei were stained with DAPI at 37 °C. Sections were mounted with buffered glycerol and observed under a fluorescence microscope for documentation.

The cells were rinsed with PBS and lysed using IP cell lysate (AWB0144, Abiowell). After centrifugation, the supernatant was incubated overnight with VPO1 or RIPK1 antibodies. To capture antigen-antibody complexes, 20 µL of protein A/G agarose was added to the lysate and incubated with the antibodies at 4 °C for 2 h with gentle shaking. Following Co-IP, the levels of VPO1 and CYLD, as well as the expression of RIPK1 and Ubiquitin (#3936, 1:1000, Boston, MA, USA), were assessed by WB.

Cardiac performance indices were calculated using Vevo software (Vevo 1100, VisualSonics, Canada). During surgery, rats were anesthetized with 2.5% isopropyl ether (80111128, Sinopharm Chemical Reagent Co., Ltd). Parameters analyzed included left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD).

Rat myocardial tissue sections were baked at 60 °C for 12 h. The sections were then immersed in xylene (10023418, Sinopharm) for 20 min, followed by sequential immersion in ethanol at concentrations of 100%, 100%, 95%, 85%, and 75% for 5 min each. Sections were stained with hematoxylin (AWI0009a, Abiowell) for 5 min, rinsed with distilled water and PBS, and then stained with eosin (AWI0032a, Abiowell) for 3 min. After dehydration with gradient alcohol (95–100%), sections were cleared in xylene for 10 min, mounted with neutral gum (G0503, Sigma, Saint Louis, MO, USA), and observed under a microscope.

Myocardial fibrosis was assessed using a Masson staining kit (AWI0253a, Abiowell). Rat myocardial tissue sections were first baked. A sufficient amount of nuclear staining solution was applied dropwise to cover the entire section, and staining was performed for 1 min. The sections were then immersed in distilled water, followed by soaking in PBS for 5–10 min to restore the blue color of the cell nuclei. Next, the plasma staining solution was applied to the sections. After staining, the solution was discarded, and the sections were treated with a re-staining solution, and then rinsed with anhydrous ethanol. Finally, the tissue sections were dried, cleared with xylene, and sealed.

Statistical analysis was performed using GraphPad Prism 8 software (8.0.2.263, San Diego, CA, USA). The data are presented as mean $\pm$ standard deviation (SD). Each experiment was performed in triplicate. Normal distribution was assessed, and comparisons between two groups were made using the T-test, while comparisons among multiple groups were conducted using One-way ANOVA. Statistical significance was set at p 0.05.

To investigate the role of VPO1 in CHF, we induced CHF in rat models using ADR. Our results demonstrated a significant upregulation of VPO1 expression in these CHF models (Fig. 1A). IHC further confirmed an elevated level of VPO1 in the Model group compared to controls (Fig. 1B). These findings suggested that VPO1 may play a role in CHF-related processes.

Fig. 1.

Vascular peroxidase 1 (VPO1) was upregulated in rats with adriamycin (ADR)-induced Chronic heart failure (CHF). (A) Expression of VPO1 was assessed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot (WB). (B) VPO1 levels confirmed by Immunohistochemistry (IHC) staining. 400× scale bar = 25 µm, 100× scale bar = 100 µm. ^*p 0.05 vs. Control. n = 5.

We also examined the expression of VPO1 in cardiomyocytes, fibroblasts, and endothelial cells derived from rat heart tissues. By using markers such as Troponin T for cardiomyocytes, vimentin for fibroblasts, and CD31 for endothelial cells [28], we performed co-localization studies. Our findings indicated that VPO1 co-localized with Troponin T, vimentin, and CD31. Moreover, CHF may affect VPO1 expression in cardiomyocytes, fibroblasts and endothelial cells, suggesting that VPO1 may also influence CHF pathology (Supplementary Fig. 1A–C).

To further explore the relationship between VPO1 and programmed necrosis in H9c2 cells, we transfected cells with VPO1-specific siRNA to silence VPO1 expression. Transfection with si-VPO1 markedly reduced VPO1 levels (Fig. 2A). ADR administration upregulated VPO1 expression, whereas si-VPO1 transfection mitigated this increase (Fig. 2B). The level of LDH, a marker of cell necrosis [29], was also measured. Knockdown of VPO1 reversed ADR-induced decreases in H9c2 cell viability and increases in LDH levels and cell necrosis (Fig. 2C–E). RIPK1, RIPK3, and MLKL are key markers of programmed cell necrosis [13]. Therefore, we assessed their relative expression and protein phosphorylation. At the mRNA level, VPO1 knockdown diminished RIPK3 and MLKL expression but had no effect on RIPK1 mRNA levels (Fig. 2F). At the protein level, VPO1 knockdown lowered the phosphorylation of RIPK1, RIPK3, and MLKL (Fig. 2G). Additionally, immunofluorescence analysis revealed a reduction in RIPK1 levels following VPO1 knockdown (Fig. 2H). These findings suggest that VPO1 downregulation alleviates programmed necrosis in H9c2 cells.

Fig. 2.

VPO1 downregulation alleviates programmed necrosis in H9c2 cells. (A) VPO1 mRNA levels were assessed by reverse transcription (RT)-qPCR. (B) VPO1 protein levels were evaluated by WB. (C) Optical density (OD) value. (D) LDH levels were measured with a kit. (E) Necrosis rate was detected by flow cytometry with Annexin V-FITC/PI staining. (F) mRNA levels of RIPK1, RIPK3, and MLKL were examined by RT-qPCR. (G) Phosphorylation of RIPK1, RIPK3, and MLKL was measured by WB. (H) RIPK1 protein levels were assessed by immunofluorescence (IF). Scale bar = 25 µm. ^*p 0.05 vs. Control. ^#p 0.05 vs. Model + si-NC. n = 3. LDH, lactate dehydrogenase; RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; MLKL, mixed lineage kinase domain-like protein; NC, negative control.

To explore the effect of VPO1 on RIPK1 ubiquitination, we employed cycloheximide (CHX) to inhibit protein synthesis, thereby promoting the gradual degradation of existing proteins and revealing their stability [27]. As exhibited in Fig. 3A, RIPK1 levels decreased progressively with increasing CHX treatment time. Notably, the knockdown of VPO1 exacerbated the reduction in RIPK1 stability induced by CHX treatment (Fig. 3A). Additionally, VPO1 knockdown led to increased ubiquitination of RIPK1 (Fig. 3B). Ubiquitination of RIPK1 is known to be regulated by deubiquitinases [30, 31, 32, 33, 34]. We measured the levels of several deubiquitinases, including CYLD [30], A20 [31], OTUD1 [32], OTUD7b [33], and OTULIN [34], using WB. However, VPO1 knockdown specifically lowered CYLD levels without affecting other deubiquitinases (Fig. 3C). Furthermore, we observed that VPO1 interacted with CYLD (Fig. 3D), suggesting that VPO1 may inhibit RIPK1 ubiquitination through CYLD. To further validate this, we overexpressed CYLD. Compared to the Model + si-VPO1 + oe-NC group, overexpression of CYLD increased both CYLD and RIPK1 levels while reducing RIPK1 ubiquitination (Fig. 3E,F). These results demonstrated that VPO1 inhibited RIPK1 ubiquitination via CYLD.

Fig. 3.

VPO1 inhibits RIPK1 ubiquitination via deubiquitinase CYLD. (A) RIPK1 protein stability was assessed by WB. (B) RIPK1 ubiquitination was examined by Co-Immunoprecipitation (Co-IP). (C) Levels of deubiquitinases CYLD, A20, OTUD1, OTUD7b, and OTULIN were measured by WB. (D) Interaction between VPO1 and CYLD was visualized by Co-IP. (E) Levels of CYLD and RIPK1 were determined by WB. (F) RIPK1 ubiquitination was calculated by Co-IP. ^*p 0.05 vs. Model + si-NC. ^#p 0.05 vs. Model + si-VPO1 + oe-NC. n = 3. oe, overexpressed.

Next, we investigated whether the alleviation of programmed necrosis in H9c2 cells by VPO1 downregulation was associated with the CYLD/RIPK1 axis. As depicted in Fig. 4A–C, overexpression of CYLD counteracted the reduction in H9c2 cell viability, lower LDH levels, and decreased cell necrosis observed with VPO1 knockdown. Moreover, in contrast to the Model + si-VPO1 + oe-NC group, CYLD overexpression enhanced the phosphorylation of RIPK1, RIPK3, and MLKL (Fig. 4D). These findings suggested that VPO1 downregulation could alleviate programmed necrosis in H9c2 cells through the CYLD/RIPK1 axis.

Fig. 4.

VPO1 downregulation alleviates programmed necrosis in H9c2 cells via the CYLD/RIPK1 axis. (A) H9c2 cell viability was measured by Cell Counting Kit-8 (CCK-8) assay. (B) LDH levels were calculated using a kit. (C) Necrosis rate was detected by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining. (D) Phosphorylation levels of RIPK1, RIPK3, and MLKL were measured by WB. ^*p 0.05 vs. Control. ^#p 0.05 vs. Model + si-NC. ^&p 0.05 vs. Model + si-VPO1 + oe-NC. n = 3.

Cellular programmed necrosis is tightly related to CHF [10]. To explore whether the downregulation of VPO1 could improve ADR-induced CHF in rats through the CYLD/RIPK1 axis, we utilized the CHF rat model for subsequent experiments. LVEF, LVFS, LVESD, and LVEDD are commonly recognized for evaluating cardiac structure and function [35]. As shown in Fig. 5A, rats in the Model group exhibited lowered LVEF and LVFS levels and elevated LVESD and LVEDD levels in contrast to the Control group, suggesting that CHF weakened heart function in rats. However, the knockdown of VPO1 attenuated the decline in cardiac function of CHF mice, and this effect could be reversed by CYLD overexpression (Fig. 5B). Additionally, CHF is often accompanied by cardiac enlargement and thickening [36]. Thus, we determined the ratios of heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL). Compared to the Control group, the ratios of HW/BW and HW/TL were raised in the Model group, suggesting that the heart was enlarged and thickened under the impact of CHF. Comparatively, the knockdown of VPO1 alleviated this phenomenon, but the role of VPO1 knockdown was inhibited by overexpression of CYLD (Fig. 5B). Next, we observed the levels of myocardial damage and fibrosis in the left ventricle of rats. Rats in the Model group had exacerbated myocardial damage and myocardial fibrosis relative to the Control group. However, VPO1 knockdown alleviated myocardial morphological damage and myocardial fibrosis and CYLD overexpression suppressed this ameliorative effect of VPO1 knockdown on myocardial damage and fibrosis (Fig. 5C,D). Moreover, we hypothesized that the levels of cardiac injury-related factors NT-proBNP, AST, LDH, and CK-MB in serum. CHF raised the levels of NT-proBNP, AST, LDH, and CK-MB, suggesting that cardiac injury was increased, and our results showed that VPO1 downregulation reduced the NT-proBNP, AST, LDH, and CK-MB levels. Overexpression of CYLD reversed the mitigating effects of VPO1 downregulation on cardiac injury (Fig. 5E). The above results postulated that downregulation of VPO1 could improve ADR-induced CHF in rats via the CYLD/RIPK1 axis.

Fig. 5.

VPO1 downregulation improves ADR-induced CHF in rats via the CYLD/RIPK1 axis. (A) Cardiac function was assessed by echocardiography measuring LVEF, LVFS, LVESD, and LVEDD. (B) Ratios of HW/BW and HW/TL were calculated. (C) Myocardial damage was evaluated using HE staining. The arrows represent the areas of myocardial injury. 400× scale bar = 25 µm, 100× scale bar = 100 µm. (D) Myocardial fibrosis was assessed by Masson staining. The arrows represent the areas of myocardial fibrosis. 400× scale bar = 25 µm, 100× scale bar = 100 µm. (E) Serum levels of NT-proBNP, AST, LDH, and CK-MB were measured. ^*p 0.05 vs. Control. ^#p 0.05 vs. Model + si-NC. ^&p 0.05 vs. Model + si-VPO1 + oe-NC. n = 5. HW/BW, weight/body weight; HW/TL, heart weight/tibia length; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter; NT-proBNP, N-terminal pro b-type natriuretic peptide; AST, Aspartate aminotransferase; CK-MB, creatine kinase-myocardial band.

We further validated whether VPO1 downregulation could improve ADR-induced programmed necrosis of rat myocardial cells through the CYLD/RIPK1 axis. VPO1 knockdown reversed the ADR-induced elevation of both VPO1 and CYLD levels. In contrast to the Model + si-VPO1 + oe-NC group, overexpression of CYLD enhanced CYLD levels without significantly altering VPO1 levels (Fig. 6A), indicating that changes in CYLD expression did not affect VPO1 levels. To assess myocardial cell necrosis, we applied WGA to localize myocardial cells and EBD to label damaged cells [37, 38]. IF analysis implicated that VPO1 downregulation lowered myocardial cell necrosis in rats, whereas CYLD overexpression reversed this effect (Fig. 6B). Additionally, CYLD overexpression negated the reductions in phosphorylation of RIPK1, RIPK3, and MLKL caused by VPO1 knockdown (Fig. 6C). These findings confirmed that VPO1 downregulation improved ADR-induced programmed necrosis of rat myocardial cells via the CYLD/RIPK1 axis.

Fig. 6.

VPO1 downregulation improves ADR-induced programmed necrosis of rat myocardial cells via the CYLD/RIPK1 axis. (A) Levels of VPO1 and CYLD were examined by WB. (B) Myocardial tissue necrosis was evaluated by immunofluorescence (IF). Scale bar = 25 µm. (C) Levels of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL were measured by WB. ^*p 0.05 vs. Control. ^#p 0.05 vs. Model + si-NC. ^&p 0.05 vs. Model + si-VPO1 + oe-NC. n = 5.